Long-term maintenance of cytochrome P450 activities by rat hepatocyte/3T3 cell co-cultures in heparinized human plasma.

Little information on the effect of plasma on hepatocyte cytochrome P450 (CYP) activities is currently available. We characterized the effect of plasma on CYPs of hepatocyte-mesenchymal cell co-cultures, which exhibit stable liver specific functions and may be potentially useful for bioartificial liver design. Rat hepatocyte-mouse 3T3-J2 cell co-cultures were maintained for 6 days in medium, and then switched to heparinized human plasma containing 3-methylcholanthrene (3MC; 2 microM), phenobarbital (PB; 1 mM), or no inducer for up to 7 days. CYP activities were measured in situ based on the o-dealkylation of ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD), or benzyloxy- (BROD) resorufin. Plasma alone increased PROD/BROD but not EROD/MROD. The endogenous inducer was in the high molecular weight fraction (>5 kD) of plasma and inhibited by >5 nM okadaic acid and >10 microM dibutyryl cyclic AMP, two inhibitors of PB-inducible CYPs. Furthermore, plasma increased CYP1A1 and CYP2B1/2 mRNA levels. In plasma, 3MC induced EROD/MROD to about 60% of the level induced in culture medium while PB induced PROD/BROD that were three- to 10-fold above levels induced in medium. CYP activities decreased between days 2 and 7 of plasma exposure, but were enhanced by plasma supplementation with amino acids, insulin, glucagon, and hydrocortisone.

Effect of flow on the detoxification function of rat hepatocytes in a bioartificial liver reactor.

Ethoxyresorufin-o-deethylation (EROD) can be used as a sensitive measure of hepatic detoxification function. In this study, we employed a fluorescence assay based on EROD to study the effect of varying Peclet number (or flow) on hepatic function in a microchannel flat-plate bioartificial liver (BAL) reactor containing a coculture of hepatocytes and fibroblasts. Static culture and reactor flow experiments established that: 1) a pseudo-steady-state detoxification rate could be attained at each Peclet number, 2) the steady-state detoxification rate increased nonlinearly with Peclet number (ranging from 167 to 2500), 3) the uptake rate of substrate was a linear function of cell surface substrate concentration (<1 microM), and 4) a shear stress of 10 dyne/cm2 did not adversely affect hepatic function for at least 12 h. A convection-diffusion-reaction model supports the conclusion that increased convective mass transfer of substrate to the cell surface is the primary cause of the observed increase in EROD rate with Peclet number. Our results suggest that detoxification rates can be enhanced by an order of magnitude by choosing an appropriate Peclet number. For our bioreactor configuration, this optimum corresponds to a Peclet number range of 1000-2000 at a Damkohler number of 0.55. The usefulness of the mathematical model is discussed in the context of scale-up to a clinical BAL reactor for human application.

Amino acid supplementation improves cell-specific functions of the rat hepatocytes exposed to human plasma.

Maintaining hepatocyte function during plasma exposure is critical for the successful development of hepatocyte-based bioartificial liver assist systems. Past attempts to culture hepatocytes in plasma yielded discouraging results. Using a stable culture model based on sandwiching hepatocytes between two layers of collagen gel, we investigated the effect of hormone and amino acid supplementation during exposure of rat hepatocytes to heparin-treated human plasma for 1 week. Morphology and hepatocyte-specific functions were evaluated for hepatocytes cultured in Dulbecco's Modified Eagle medium (DMEM), nonsupplemented plasma, plasma supplemented with hormones, or with hormones plus amino acids. Amino acids were supplemented at four-fold concentration of Basal Medium Eagle with 4 mM glutamine, whereas hormones included 7.5 microg/mL of hydrocortisone and 50 microU/mL of insulin. Cuboidal structure and bile canaliculi formation were observed throughout the 1-week exposure period for control hepatocytes in DMEM and for hepatocytes cultured in hormone supplemented plasma. Albumin and urea synthesis rates of hepatocytes in hormone plus amino acid supplemented plasma during the last day of plasma exposure were 60.4 +/- 13.7 and 75.6 +/- 6.5 (microg/day per 1 x 10(6) cells, mean +/- SD), respectively, comparable to cultures in standard culture medium. On the other hand, hepatocytes exposed to nonsupplemented plasma suffered significant morphological and functional damage. The results of this study indicate that hormone plus amino acid supplementation help to restore function in hepatocytes exposed to plasma.

Optimization of rat hepatocyte culture in citrated human plasma.

BACKGROUND: Maintenance of liver-specific functions in hepatocyte cultures during plasma exposure is critical for the clinical application of bioartificial liver assist systems. Sodium citrate is a common anticoagulant but has been shown to be cytotoxic to hepatocytes. We have tested the effect of various supplements on the viability and function of adult primary rat hepatocytes exposed to citrated plasma. MATERIALS AND METHODS: Freshly isolated rat hepatocytes were cultured in the collagen gel sandwich configuration in culture medium for 6 days followed by exposure to citrated human plasma with various supplements for 1 week. Controls were left in culture medium throughout. Viability and synthetic functions were evaluated. RESULTS: Hepatocytes exposed to unsupplemented citrated plasma lost significant viability and function within the first 2 days. Cells cultured in plasma supplemented with a fivefold concentrate of standard hepatocyte culture medium maintained urea (1. 2-2.1 micromol/day/10(6) cells) and albumin (51-62 microg/day/10(6) cells) synthesis rates equal to or higher than those of controls. Among the various components of the concentrated medium supplement, calcium chloride (1.8 mM), magnesium sulfate (0.8 mM), amino acids (fourfold Basal Medium Eagle amino acids including 4 mM glutamine), and glucagon (14 ng/ml) were found to be essential in maintaining urea synthesis. Maintenance of a high albumin synthesis rate also required the addition of hydrocortisone (7.5 microg/ml) and insulin (0.5 U/ml). CONCLUSIONS: Appropriate metabolic and hormonal supplementation of citrated human plasma prevents its cytotoxic effects and may be used in conjunction with in vivo use of bioartificial liver assist systems.

Expression of toll-like receptor 2 on gamma delta T cells bearing invariant V gamma 6/V delta 1 induced by Escherichia coli infection in mice.

We recently reported that the number of gamma delta T cells was increased after infection with Escherichia coli in C3H/HeN mice. We here showed that an i.p. injection with native lipid A derived from E. coli induced an increase of gamma delta T cells in the peritoneal cavity of LPS-responsive C3H/HeN mice and, albeit to a lesser degree, also in LPS-hyporesponsive C3H/HeJ mice. The purified gamma delta T cells from C3H/HeN and C3H/HeJ mice expressed a canonical TCR repertoire encoded by V gamma 6-J gamma 1/V delta 1-D delta 2-J delta 2 gene segments and proliferated in response to the native lipid A derived from E. coli in a TCR-independent manner. The lipid A-reactive gamma delta T cells bearing canonical V gamma 6/V delta 1 expressed Toll-like receptor (TLR) 2 mRNA, while TLR4 mRNA was undetectable. Treatment with a TLR2 anti-sense oligonucleotide resulted in hyporesponsiveness of the gamma delta T cells to the native lipid A. TLR2-deficient mice showed an impaired increase of the gamma delta T cells following injection of native lipid A. These results suggest that TLR2 is involved in the activation of canonical V gamma 6/V delta 1 T cells by native E. coli lipid A.

Prostaglandin E(1) protects against liver injury induced by Escherichia coli infection via a dominant Th2-like response of liver T cells in mice.

Prostaglandin E series (PGEs) are known to protect against lipopolysaccharide (LPS)-induced liver injury by down-regulating the production of inflammatory cytokines. We show here a novel mechanism whereby prostaglandin E(1) protects mice against liver injury after Escherichia coli infection. Prostaglandin E(1) administration suppressed circulating interleukin 12 (IL-12) levels but increased the IL-10 production after E. coli challenge. Furthermore, prostaglandin E(1)-alpha-cyclodextrin (PGE(1)) shifted the Th1/Th2 balance of CD3(intermediate) IL-2Rbeta(+) T cells in the liver to a dominant Th2-like response. Neutralization of endogenous IL-4 by administration of anti-IL-4 monoclonal antibody (mAb) diminished the inhibitory effect of prostaglandin E(1) on liver injury after E. coli challenge. These results suggested that the Th2-like response of liver T cells may be at least partly involved in the mechanism whereby prostaglandin E(1) protects against E. coli-induced liver injury.

Interleukin-15 production at the early stage after oral infection with Listeria monocytogenes in mice.

We previously reported that exogenous interleukin-15 (IL-15) induces proliferation and activation of intestinal intraepithelial lymphocytes (i-IEL) in naive mice. To investigate the ability of endogenous IL-15 to stimulate i-IEL in vivo, we monitored i-IEL and intestinal epithelial cells (i-EC) in mice after an oral infection with Listeria monocytogenes. Although the populations of alphabeta and gammadelta i-IEL were not significantly changed after the oral infection, the expression level of interferon-gamma (IFN-gamma) was increased both at transcriptional and protein levels, and a conversely marked decrease in interleukin-4 (IL-4) was detected in the i-IEL on day 1 after infection as compared with before infection. The T helper 1 (Th1)-biased response of i-IEL coincided with a peak response of IL-15 production in the i-EC after oral infection. These results suggested that IL-15 produced from i-EC may be at least partly involved in the stimulation of i-IEL to produce IFN-gamma after oral infection with L. monocytogenes.

MHC class II-dependent NK1.1+ gammadelta T cells are induced in mice by Salmonella infection.

We observed the emergence of a novel population of gammadelta T cells expressing NK1.1 Ag in the peritoneal cavity of mice infected with Salmonella choleraesuis. The NK1.1+gammadelta T cells accounted for approximately 20% of all gammadelta T cells emerging in the peritoneal cavity of C57BL/6 mice and expressed preferentially rearranged Vgamma4-Jgamma1 and Vdelta6.3-Ddelta1-Ddelta2-Jdelta1 genes with N diversity. The gammadelta T cells proliferated vigorously in response to PHA-treated spleen cells and produced IFN-gamma in the culture supernatant. However, spleen cells from Abetab-deficient mice were unable to stimulate the gammadelta T cells. Furthermore, the NK1.1+gammadelta T cells were stimulated not only by Chinese hamster ovary (CHO) cells expressing wild-type IAb but also by those expressing IAb/Ealpha52-68 or IAb/pigeon cytochrome c-derived analogue peptide complex. These proliferation activities were inhibited by mAb specific for IAb chain. Consistent with these findings, the emergence of NK1.1+gammadelta T cells was reduced in the peritoneal cavity of Abetab-deficient mice after Salmonella infection, whereas NK1.1+gammadelta T cells were rather abundant in the peritoneal cavity of Salmonella-infected beta2m-deficient mice. Moreover, the NK1.1+gammadelta T cells were easily identified in the thymus of beta2m-deficient but not Abetab-deficient mice. Our results indicated that MHC class II expression is essential for development and activation of NK1. 1+gammadelta T cells in the thymus and the periphery.

Interleukin-15 may be responsible for early activation of intestinal intraepithelial lymphocytes after oral infection with Listeria monocytogenes in rats.

Exogenous interleukin-15 (IL-15) stimulates intestinal intraepithelial lymphocytes (i-IEL) from mice to proliferate and produce gamma interferon (IFN-gamma) in vitro. To determine whether endogenous IL-15 is involved in activation of i-IEL during intestinal infection, we examined IL-15 synthesis by intestinal epithelial cells (i-EC) after infection with Listeria monocytogenes in rats. In in vitro experiments, invasion of L. monocytogenes into IEC-6 cells, a rat small intestine epithelial cell line, evidently induced IL-15 mRNA expression coincident with nuclear factor kappaB (NF-kappaB) activation, which is essential for IL-15 gene expression. IL-15 synthesis was detected in rat i-EC on day 1 after an oral inoculation of L. monocytogenes in vivo. The numbers of T-cell receptor (TCR) gamma delta+ T cells, NKR.P1(+) cells, and CD3(+) CD8(+) alpha alpha cells in i-IEL were significantly increased on day 1 after oral infection. The i-IEL from infected rats produced larger amounts of IFN-gamma upon stimulation with immobilized anti-TCR gamma delta or anti-NKR.P1 monoclonal antibodies. These results suggest that IL-15 produced by i-EC may stimulate significant fractions of i-IEL to produce IFN-gamma at an early phase of oral infection with L. monocytogenes.

Prostaglandin E2 protects against liver injury after Escherichia coli infection but hampers the resolution of the infection in mice.

cAMP-increasing agents such as prostaglandin E2 (PGE2) are known to protect against LPS-induced liver injury by downregulating the production of inflammatory cytokines such as TNF-alpha. However, the effects of such reagents on host defense against bacterial infection remain unknown. We show here that in vivo administration of PGE2 significantly protected mice against liver injury after Escherichia coli infection but hampered the resolution of the infection. PGE2 significantly suppressed circulating TNF-alpha and IL-12 levels but increased the IL-10 production after E. coli challenge. PGE2 inhibited the emergence of gammadelta T cells in the peritoneal cavity, which are important for host defense against E. coli, and deteriorated bacterial exclusion in the peritoneal cavity after E. coli challenge. These results suggested that PGE2 affects host defense mechanisms against E. coli infection through modulation of cytokine production and gammadelta T cell accumulation.

Protective roles of gamma delta T cells and interleukin-15 in Escherichia coli infection in mice.

The number of gamma delta T cells in the peritoneal cavity was increased after an intraperitoneal (i.p.) infection with Escherichia coli in lipopolysaccharide (LPS)-responsive C3H/HeN mice but not in LPS-hyporesponsive C3H/HeJ mice. The gamma delta T cells preferentially expressed invariant Vgamma6 and Vdelta1 chains and proliferated to produce a large amount of gamma interferon in the presence of LPS. Mice depleted of gamma delta T cells by T-cell receptor delta gene mutation showed impaired resistance against E. coli as assessed by bacterial growth. Macrophages from C3H/HeN mice infected with E. coli expressed higher levels of interleukin-15 (IL-15) mRNA than those from the infected C3H/HeJ mice. Administration of anti-IL-15 monoclonal antibody inhibited, albeit partially, the appearance of gamma delta T cells in C3H/HeN mice after E. coli infection and diminished the host defense against the infection. These results suggest that LPS-stimulated gamma delta T cells play an important role in the host defense against E. coli infection and that IL-15 may be partly involved in the protection via an increase in the gamma delta T cells.

The NF-kappaB binding site is essential for transcriptional activation of the IL-15 gene.

We cloned the 5' upstream region of IL-15 genomic DNA and examined promoter activity in macrophages stimulated with lipopolysaccharide(LPS). The 1.2 kilobase (kb) fragment of the 5' upstream region contained binding elements for LPS-inducible transcription factors such as NFIL-6 or NF-kappaB. Determined by luciferase assay following transient transfection in the J774A.1 macrophage cell line, the 1.2 kb of the 5' upstream region exhibited high promoter activity in response to LPS, while promoter activity was significantly reduced by the 5' deletion of 313 base pairs containing the NF-kappaB binding motif. Nuclear protein prepared from LPS-stimulated macrophages formed a complex with the NF-kappaB binding sequence of the IL-15 promoter. Taken together, the binding of nuclear protein to the NF-kappaB binding site is required for transcriptional activation of the IL-15 gene in LPS-stimulated macrophages.

Translational efficiency is up-regulated by alternative exon in murine IL-15 mRNA.

IL-15 promotes the growth of T cells and shares properties of IL-2. IL-2 is produced exclusively by T cells, while IL-15 message is expressed by a variety of tissues. However, it has been difficult to demonstrate IL-15 in the supernatants of many cells that express message for this cytokine. This suggests that IL-15 production is regulated by post-transcriptional controls. In this study, we cloned three types of murine IL-15 cDNA isoforms generated by alternative splicing and compared the translational efficiency among these isoforms. The translational efficiency of isoforms with alternative exon 5 containing another 3' splice site was significantly higher than that of IL-15 cDNA with originally described exon 5, which is generated by internal splicing of alternative exon 5. The translation product of the isoform containing alternative exon 5 has a shorter open reading frame due to stop codons in additional sequence, followed by a new AUG codon, and displays a shorter leader sequence. The shorter isoform of the IL-15 was detected in peritoneal macrophages stimulated with IFN-gamma and LPS, which expressed an abundant level of alternative exon 5. These results suggest that normal IL-15 production in stimulated macrophages is regulated by splicing of alternative exon 5.